Amplification and detection of nucleic acids

ABSTRACT

The present disclosure relates to a sample assessment device. By way of example, the sample assessment device may include a substrate including a sample application region; an amplification region comprising a plurality of amplification reagents; a waste region comprising an entrance fluidically coupled to the amplification region and extending away from the amplification region; and a detection region spaced apart from the amplification region. The sample assessment device may also include a valve coupled to the substrate and configured to separate the amplification region from the detection region in a closed configuration, wherein the amplification region and the valve are positioned on the sample assessment device between the sample application region and the detection region and wherein the sample assessment device is configured to permit lateral flow from the amplification region to the detection region when the valve is in an open configuration.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH & DEVELOPMENT

This invention was made with Government support under contract numberHR0011-11-C-0127 awarded by DARPA. The Government has certain rights inthe invention.

BACKGROUND

The subject matter disclosed herein relates to amplification anddetection of nucleic acids using lateral flow devices.

Caregivers may use diagnostic tests to determine if a patient has aparticular clinical condition. Such tests may be performed by testing apatient sample (e.g., blood or urine) for the presence of one or moremarkers such as proteins or small molecules and, depending on theircomplexity, the tests may be performed in a dedicated testing laboratoryor at the point of care, e.g., in the doctor's office or in the field.However, in certain circumstances, a diagnostic test may be a test forthe presence of a particular nucleic acid sequence, either sequences inthe patient's own genetic material or sequences associated withpathogenic infection. Relative to proteins or small molecule markers, agiven nucleic acid sequence may be present in relatively lowconcentrations in a given biological sample. Accordingly, manytechniques for assessing the presence or concentration of a nucleic acidsequence of interest rely on amplification techniques that enrich thesample by amplifying the sequence of interest prior to detection.However, the sample may include inhibitory components and/or inhibitorycomponents may be generated in upstream processes (e.g., cell lysis)that could interfere with subsequent steps such as amplification ordetection. For example, if the sample is lysed, lysis enzymes present inthe amplified sample may degrade the amplification reagents or otherwiseinterfere with the amplification or detection reactions, decreasingsensitivity to the sequence of interest. Further, the debris from celllysis (including formerly intracellular enzymes) may also degradeamplification reagents, detection reagents and/or the amplificationproduct. Certain techniques include dedicated wash steps that remove thelysis reagents and/or waste products, but washing introduces additionalcomplexity and often user intervention.

BRIEF DESCRIPTION

In one embodiment, a method of assessing a sample is provided. In oneembodiment, the method includes providing a sample assessment deviceincluding a sample application region comprising a plurality of lysisreagents; an amplification region capable of being heated to atemperature range sufficient for nucleic acid amplification; a wasteregion comprising an entrance coupled to the amplification region andextending away from the amplification region; a detection region spacedapart from the amplification region; and a valve separating theamplification region from the detection region. The method also includesapplying a sample comprising nucleic acids to the sample applicationregion; allowing the sample to react with the lysis reagents in a lysisreaction; allowing the sample to flow into the amplification region andreact with amplification reagents for a duration of time in anamplification reaction to generate amplification products of a nucleicacid sequence of interest; allowing waste from the lysis reaction toflow into the waste region; and allowing the amplification products toflow through the valve to enter the detection region, wherein the valveis configured to be closed during the amplification reaction and openafter the duration of time.

In another embodiment, a sample assessment device is provided. Thesample assessment device includes a substrate including a sampleapplication region; an amplification region; a waste region comprisingan entrance fluidically coupled to the amplification region andextending away from the amplification region; and a detection regionspaced apart from the amplification region. The sample assessment devicealso includes a valve coupled to the substrate and configured toseparate the amplification region from the detection region in a closedconfiguration, wherein the amplification region and the valve arepositioned on the sample assessment device between the sampleapplication region and the detection region and wherein the sampleassessment device is configured to permit lateral flow from theamplification region to the detection region when the valve is in anopen configuration.

In another embodiment, a sample assessment system is provided. Thesystem includes a substrate including a first region comprising aplurality of lysis reagents; a second region comprising an entrancefluidically coupled to the first region and extending away from thefirst region; and a third region spaced apart from the first region. Thesystem also includes a valve separating the first region from the thirdregion in a closed configuration to prevent lateral flow from the firstregion to the third region and configured to permit the lateral flow inan open configuration. The system also includes a detector configured todetect one or more signal molecules in the substrate and provide anoutput and a processor configured to receive the output and determine apresence or concentration of a nucleic acid sequence of interest in thebiological sample based on the output.

BRIEF DESCRIPTION OF THE DRAWINGS

These and other features, aspects, and advantages of the presentinvention will become better understood when the following detaileddescription is read with reference to the accompanying drawings in whichlike characters represent like parts throughout the drawings, wherein:

FIG. 1 is a schematic view of sample flow through a sample assessmentdevice according to an embodiment of the disclosure;

FIG. 2 is a top view of a sample assessment device according to anembodiment of the disclosure;

FIG. 3 is a cross-sectional view of the sample assessment device of FIG.2;

FIG. 4 is a top view and detail cross-sectional view of a sampleassessment device according to an embodiment of the disclosure;

FIG. 5 is a top view and detail cross-sectional view of a sampleassessment device according to an embodiment of the disclosure;

FIG. 6 is a top view of sample assessment devices including parallelcapillary sample loading according to an embodiment of the disclosure;

FIG. 7 is a cross-sectional view of an example of capillary loading of asample from a swab onto a sample assessment device according to anembodiment of the disclosure;

FIG. 8 is a cross-sectional view of an example of capillary loading of ablood sample onto a sample assessment device according to an embodimentof the disclosure;

FIG. 9 is a flow diagram of a method of assessing a sample according toan embodiment of the disclosure; and

FIG. 10 is an illustration of a sample assessment system according to anembodiment of the disclosure.

DETAILED DESCRIPTION

Paper or porous substrate-based diagnostics are of growing interest tothose that design, sell, and use point-of-care platforms. Suchdiagnostics may be low-cost, portable, and easy to use. For example,paper diagnostics have been used to detect proteins or small molecules.Implementation of paper diagnostic techniques for nucleic acidamplification is more complex. Certain techniques may involveamplification via a paper platform, but a separate end-point detectionof the amplified products, e.g., by flowing the amplified material overa capture line. Accordingly, such techniques involve more steps and usereffort relative to a paper diagnostics used for protein detection.

In one embodiment, a sample applied to a sample assessment device asprovided herein flows through an amplification region of the device(e.g., flow through a porous substrate to encounter amplificationreagents disposed in the substrate). The amplification reagents andprimers specific to a target sequence of interest to produceamplification products having, or complementary to, the sequence ofinterest. Waste products from a sample lysis process that occurs priorto amplification are displaced downstream in the lateral flow asamplification products are generated. Accordingly, in one embodiment,waste products flow through the substrate ahead of the desiredamplification products.

The sample assessment device as provided includes a diverted waste flowpath for waste to decrease one or more undesired contaminants in thesample and/or in the device itself. The waste, e.g., from lysis, flowsahead of the amplification products into the waste flow path and out ofthe main flow path of the sample assessment device. Further, the wasteis prevented from flowing downstream in the main flow path by a valvethat separates downstream portions of the sample assessment device fromthe upstream amplification region while the amplification reactionprogresses. Accordingly, the waste flow path is enriched in wasteproducts. In one embodiment, by allowing waste to flow out of theamplification region of the sample assessment device, amplification mayoccur without being hindered by lysis reagents or products that mayreduce the generation of amplicon or amplification products and/or maydegrade such products themselves. As amplification progresses,additional amplicons are generated upstream in the main flow path asadditional downstream waste is diverted into the waste flow path. Suchan implementation is in contrast to techniques that do not provide anexit path for lysis products, but instead rely of wash steps to removethese products. After a suitable time period/flow volume has elapsed topermit the majority of lysis products to flow into the waste flow pathand to permit sufficient amplification, the valve may be opened topermit lateral flow from the amplification reaction downstream towards adetection region. The products entering the detection region may includea reduced concentration of undesired waste contaminants, which in turnmay yield improved detection and reduced signal interference.

Provided herein are techniques that may be used to amplify and detectnucleic acids in a biological sample using a lateral flow solidsubstrate sample assessment device. The disclosed embodiments may beused to detect a presence of a nucleic acid sequence of interest in abiological sample. The present techniques provide amplification anddetection in a single substrate and reduced presence of contaminants inthe amplification and detection reactions. Accordingly, amplificationand detection as provided may be faster and more convenient (e.g., mayinclude no washing steps to remove contaminants) and, thus, may beperformed at the point of care or may improve workflow in a dedicatedtesting laboratory.

FIG. 1 is a schematic view of sample flow through a sample assessmentdevice 10 implemented on a substrate 12 that includes a first region 14and a second region 16 separated by a valve 20. After a lysis step(shown as step 1), a subsequent amplification step is initiated (step2). The lysis waste products flow through the sample assessment deviceahead of, or downstream of, the amplification products, as shown.

The waste products may include lysis reagents (small molecules,surfactants, membrane disruptors, lytic enzymes, etc.), the cell debrisfrom lysis (lipids, proteins, enzymes including proteases and nucleases,and other small molecules) as well as the non-cellular components of thematrix in which the cells were delivered to the device (e.g., forblood—serum proteins and proteases, platelets, hemoglobin, etc.; orother sample matrices including sputum, or environmental contaminationfrom forensic samples). Rather than being removed or neutralized throughmanual processing (column chromatography, centrifugation, washing,etc.), in the disclosed techniques, all of that separation must be donewithin the device without user intervention.

While the valve is closed, the waste flows into a waste region 28, asshown in step 3, by fluid displacement. The lysis waste is generatedprior to amplification, as the nucleic acids of interest are notamplifiable until after release from the cells and the lysis reagentsmay suppress amplification. The waste is separated from theamplification reaction, and its products, via spatial/temporalfractionation of the total reaction volume. The leading fluid fractionis where lysis occurs and contains the bulk of the soluble wasteproducts (with very little of the amplification product) and can thus bedelivered to the waste pad downstream without significant loss ofsensitivity. The fluid in the amplification zone can then amplify “inplace” (after the amplification zone and waste zone are full of liquid)until all of the amplification reagents have been used up. The wastepad, when full, can then be disconnected (step 4) and the detectionfluid path can be connected, or just the latter can occur, such that allsubsequent fluid fractions that flow through to detection (step 5)contain a minimum of waste and a maximum of amplified material.

FIG. 2 is a top view of an example of the sample assessment device 10including the substrate 12, e.g., a porous substrate, configured topermit lateral flow through the substrate 12. The first region 14 andthe second region 16 are fluidically separated by a valve 20 in a closedconfiguration. That is, fluids applied to the substrate 12 cannot flowfrom the first region 14 to the second region 16 when the valve 20 isclosed. In operation, a sample is applied to a sample application region24 and flows in the direction of arrow 25 towards an amplificationregion 26, which are in the first region 14 of the substrate 12. In someembodiments, the sample application region 24 may be overlapping with orcombined with the amplification region 26. That is, the sampleapplication region 24 may have 0%, 25%, 50% or 100% overlap with theamplification region 26 in certain embodiments. In one embodiment, thesubstrate 12 is wet prior to or is wet concurrently with sampleapplication to promote flow through the device 10. The substrate 12 alsoincludes a waste region 28 that is fluidically coupled to the firstregion 14. When the valve 20 is closed, lateral flow to downstreamdetection region 30 is prevented, and products or compounds in thesubstrate 12 may flow into the waste region via a flow entrance 48.

The substrate 12 may include, but is not limited to, materials such ascellulose, cellulose acetate, regenerated cellulose, nitrocellulose,functionalized nitrocellulose, glass fibers, quartz fibers, polymerfibers or combinations thereof. In one embodiment, the substratecomprises cellulose. In one or more embodiments, the substrate isselected from a nitrocellulose membrane, a cellulose membrane, acellulose acetate membrane, a regenerated cellulose membrane, anitrocellulose mixed ester membrane, a polyethersulfone membrane, anylon membrane, a polyolefin membrane, a polyester membrane, apolycarbonate membrane, a polypropylene membrane, a polyvinylidenedifluoride membrane, a polyethylene membrane, a polystyrene membrane, apolyurethane membrane, a polyphenylene oxide membrane, apoly(tetrafluoroethylene-co-hexafluoropropylene) membrane, and anycombination of two or more of the above membranes. In some embodiments,the substrate comprises modified matrices, such as pegylated cellulose,pegylated glass fiber or pegylated nitrocellulose. In some embodiments,the substrate is a porous substrate. In one embodiment, the substrate isa porous cellulose membrane. In one embodiment, the solid substrate is aporous cellulose paper, such as a cellulose substrate from GE HealthcareLife Sciences (formerly Whatman™). In one example, the cellulosesubstrate comprises 903-cellulose, FTA™ or FTA™ Elute. In someembodiments, the substrate 12 is a solid substrate, which is a non-waterdissolvable material, which enables collection, extraction, separation,capture, detection and storage of nucleic acids followed by elutionwithout solubilizing the material using water or aqueous buffer.

The substrate 12 may include a single layer or a plurality of layersthat are joined together or otherwise coupled. In one embodiment,adjacent functional regions (e.g., the sample application region 24, theamplification region 26) may be formed from different layers that abutor overlap to fluidically couple the adjacent layers and permit lateralflow.

A “sample” or “biological sample” may include samples taken frombiological subject, including biological tissue or fluid obtained from asubject. Such samples can be, but are not limited to, body fluid (e.g.,blood, blood plasma, serum, sweat, wound exudate or urine), organs,tissues, biopsies, fractions, and cells isolated from, or located in,any biological system, such as mammals. Biological samples also mayinclude sections of the biological sample including tissues (e.g.,sectional portions of an organ or tissue). Biological samples may alsoinclude extracts from a biological sample, for example, nucleic acidspurified from a biological fluid (e.g., blood or urine). Samples mayalso include unprocessed bodily fluids. For example, in one embodiment,it is envisioned that unprocessed bodily fluids may be applied directlyto the porous substrate without any processing. In addition, thetechniques may also be used to assess the presence of nucleic acids ofinterest in environmental samples, including forensic or securityscreening samples. In one embodiment, the techniques may be used todetermine if a particular environment (e.g., a filtering system, apiping system) has been contaminated with one or more pathogens.

Although the disclosed embodiments are generally disclosed in thecontext of nucleic acids, it should be understood that otherself-amplifying components may be tested as provided herein. Further,the nucleic acids disclosed herein may include DNA or RNA, and may be inthe form of purified nucleic acids, unprocessed biological fluids thatinclude nucleic acids or partially processed biological fluids orsamples, bacterial samples, pathogenic samples, environmental samples,etc. Nucleic acids may include all forms of DNA (e.g. genomic DNA,mtDNA) or RNA (mRNA, tRNA, rRNA, small RNA, siRNA, miRNA, non-codingRNA, animal RNA, plant RNA, viral RNA or bacterial RNA), as well asrecombinant RNA and DNA molecules or analogues of DNA or RNA generatedusing nucleotide analogues. The nucleic acids may be single stranded ordouble stranded. The nucleic acids may include the coding or non-codingstrands. Nucleic acids may also include fragments of nucleic acids, suchas naturally occurring RNA or DNA which may be recovered using theextraction methods disclosed. Nucleic acid may also refer to a portionof a nucleic acid (e.g., RNA or DNA). The extracted nucleic acids mayfurther comprise peptide nucleic acids (PNA).

In some embodiments the sample application region 24 may includeimpregnated reagents, such as stabilizing reagents or cell lysisreagents. For example, the lysis reagents may lyse cells within thesample to release genomic DNA or other nucleic acid template materials.The lysis reagents, upon reaction with the sample, may generate certainwaste products, such as proteins, lipids and small molecules. Inaddition, after the lysis is complete, the lysis enzymes themselves maybe considered waste products. The lysis reagents may include, but arenot limited to, guanidinium hydrochloride, guanidinium chloride,guanidinium isothiocyanate/thiocyanate, sodium thiocyanate, sodiumperchlorate, sodium iodide, potassium iodide, urea, proteolytic enzymes,surfactants/membrane disruptors and/or any combination thereof. Atypical anionic chaotropic series, shown in order of decreasingchaotropic strength, includes: CCl₃COO⁻, CNS⁻, CF₃COO⁻, ClO₄ ⁻, I⁻,CH₃COO⁻, Br⁻, Cl⁻, or CHO₂ ⁻. The lysis reagent may include chaotropicsubstances in concentrations of from 0.1 M to 10 M, or from 1 M to 10 M.The lysis reagents may be present in the substrate 12 prior to sampleapplication (e.g., may be impregnated in the substrate 12) or may beapplied to the substrate 12. In certain embodiments, the lysis reagentsmay be applied concurrently with the biological sample or in a separatestep.

For bacterial samples, the lysis reagents may include, for example,lytic enzymes or the biological samples may be pretreated, for example,with lytic enzymes, prior to being lysed. The lysis reagent may alsocomprise protease, such as serine, cysteine and metallic proteases. Aprotease free of nuclease may be used. A protease comprising astabilizer, such as metallic ions, may be used. The protease may beused, upon addition, in an amount of preferably from about 0.001 IU toabout 10 IU, more preferably from about 0.01 IU to about 1 IU, per ml ofthe whole lysis reagent. In some embodiments, the lysis reagent alsoincludes a sufficient amount of buffer. The examples of buffers for usein the lysis reagent include tris-(hydroxymethyl) aminomethanehydrochloride (Tris-HCl), sodium phosphate, sodium acetate, sodiumtetraborate-boric acid and glycine-sodium hydroxide. In someembodiments, the lysis reagent also includes a non-ionic surfactant, acationic surfactant, an anionic surfactant, an amphoteric surfactant,and/or any combination thereof. Exemplary nonionic surfactants include,but are not limited to, t-octylphenoxypolyethoxyethanol (TRITON X-100™),(octylphenoxy)polyethoxyethanol (IGEPAL™ CA-630/NP-40),triethyleneglycol monolauryl ether (BRIJ™ 30), sorbitari monolaurate(SPAN™ 20), or the polysorbate family of chemicals, such as polysorbate20 (i.e., TWEEN™ 20), TWEEN™ 40, TWEEN™ 60 and TWEEN™ 80 (Sigma-Aldrich,St. Louis, Mo.). Examples of cationic surfactants includecetyltrimethylammonium bromide, dodecyltrimethylammonium chloride,tetradecyltrimethylammonium chloride and cetylpyridinium chloride. Theconcentration of the surfactant in the lysis reagent could vary slightlyamong the different surfactants and depending on the components in thebiological sample to be lysed. In some embodiments, the concentration ofthe surfactant is in a range of from about 0.01% to about 20% by weight.The lysis reagent may further comprise dithiothreitol (DTT).

The substrate 12 may also be provided with amplification reagents,including one or more primers, i.e., oligonucleotides complementary to atarget sequence of interest and/or capable of hybridizing to a targetsequence of interest. Sequences of interest may include sequencesassociated with particular pathogens, particular diseases, or particularmutations (e.g., mutations which confer disease resistance). Further,the primers may include multiple primers complementary to differentregions of a single desired sequence. The amplification reagents mayalso include one or more polymerases and nucleotides as well as one ormore appropriate buffers. The amplification reagents may be selected towork in conjunction with a particular amplification technique (e.g.,loop-mediated isothermal amplification, strand displacementamplification, helicase dependent amplification, nicking enzymeamplification reaction). In one embodiment, the amplification reagentsmay be selected to be used in isothermal or polymerase chain reactionamplification.

In one embodiment, the substrate 12 may be provided with theamplification reagents distributed throughout the amplification region26. In another embodiment, the substrate 12 may be provided with theamplification reagents dried on the substrate 12. An end user may wetthe substrate 12 prior to use by exposing the substrate 12 to anappropriate buffer. In another embodiment, the substrate 12 may beprovided with separate amplification reagents that the end user mayapply to the substrate 12. The amplification reagents may be provided asseparate components or as a premixed solution as part of a kit includingone or more sample assessment devices.

In some applications in which the end user desires a substrate 12 fortesting a particular target sequence, the primers may be provided aspart of the amplification kit. That is, the amplification kit may beprovided with all of the amplification reagents, including primers for aparticular target sequence. An end user may purchase the amplificationkit that corresponds with a particular disease, pathogen or clinicalcondition. In other applications, the primers may be provided separatelyor provided by the end user. For example, it is envisioned that thesubstrate 12 may be used to assess any target sequence desired by theend user. To that end, the end user may select an appropriate primer setfor application to the porous substrate. In such embodiments, the enduser may then apply the desired primers to the substrate 12.

In one embodiment, the waste region 28 may be one or more separatelayers 50 of the substrate 12, as shown in the cross-sectional view ofthe sample assessment device 10 in FIG. 3, in direct contact with theamplification region 26 to fluidically couple the amplification region26 to the waste region to facilitate lateral flow in the direction ofarrow 52 and into the waste region 28. In another embodiment, the wasteregion 28 may be formed as an extending portion of the amplificationregion 26, e.g., the waste region 28 may be integral with theamplification region 26. When the valve 20 is closed flow in thedirection of arrow 56 terminates before the detection region 30, whichis in the second region 16. As shown in FIG. 3, in one embodiment, thefirst region 14 and the second region 16 are formed from a first layer57 and a second layer 58 of the substrate 12 and separated by the valve20 to prevent flow from the first region 14 into the second region 16,i.e., to prevent flow into the detection region 30.

In certain embodiments of the disclosure, the valve 20 may be amicromechanical valve, such as a check valve. In another embodiment, thevalve 20 may be a hydrogel valve. In one embodiment, the hydrogel valveis configured to melt or dissolve upon exposure to a chemical (e.g.,glucose concentration) or environmental condition (temperature or pHchange, exposure to an electrical or magnetic field). Once melted ordissolved, the valve 20 is irreversibly positioned in the openconfiguration. In another embodiment, the exposure may change a porosityof the hydrogel to a more porous configuration to permit flow, whereby aless porous configuration prevented flow in the closed configuration.

The sample assessment device 10 may also include protective layers 60and 62, between which the substrate 12 is positioned. To permit sampleapplication, a portion of the substrate 12 (e.g., portion 64), may beextended beyond the protective layers 60 and 62 or may be otherwiseaccessible, e.g., via a hole or passage. When present, the protectivelayers 60 and 62 may be formed of generally transparent or translucentmaterials that permit detection in the detection zone of amplifiedproducts via optical or other techniques, as discussed herein.

In some embodiments, the substrate 12 includes an elongated strip alongthe main flow path and on which the sample application region 24, theamplification region 26, and the detection region 30 are all disposed.The waste region 28 may extend away from the elongated strip. The runtime starting from sample application to capture of target nucleic acidmay increase with increasing the length of the target nucleic acid, asthe high molecular weight target nucleic acids have low mobility andmigrate slowly. The separation of the nucleic acids from the sample mayimprove with increasing length of the elongated strip of the substrate12. The length of the elongated strip may be optimized consideringbetter separation as well as run time. The elongated strip may have alength in a range between 1 cm and 20 cm. In some embodiments, theelongated strip has a length less than 10 cm.

FIG. 4 is an embodiment of the sample assessment device 10 in which thevalve 20 is triggered to open via flow into the waste region 28. Anin-line valve 20 may provide the benefit of less complex manufacturing,because the valve 20 may be relatively simple. The sample, when applied,flows in the direction of arrow 70 through the sample application region24, which may include lysis reagents provided as a pad or layer 72 ofthe substrate 12. When the sample flows into the sample applicationregion 24, the reagents in the pad or layer, which is fluidicallycoupled to the sample application region, contact the sample. In thismanner, a substrate 12 may be formed from less expensive materials andmay include small areas of more expensive materials that are configuredto include reagents as well as appropriate stabilizing or preservativematerials.

Amplified products of interest are generated in the amplification region26 and displace waste that enters the waste region 28 via entrance 48 ora portion of the waste region 28 fluidically coupled to the waste region28 prior to the amplification flow. The waste region 28, in turn,includes a flow path that is sized and shaped so that a time to flow aterminus 76 is sufficient to permit adequate amplification. Accordingly,the waste region may include a meandering or curved path. For example,in the depicted embodiment, the waste region 28 includes a plurality oforthogonal sections 78 (i.e., orthogonal to the main flow axis along thearrow 70) and a plurality of parallel sections 80 (i.e., parallel to themain flow axis along the arrow 70). The total length of the path may bedesigned to permit flow of a desired duration of time to the terminus76, with a longer path being associated with a longer duration of time.In addition, the total surface area of the waste region 28 may also bedesigned to permit flow of a desired duration of time to the terminus76, with a greater surface area being associated with a longer durationof time. In one embodiment, a total surface area of the waste region 28may be greater than a total surface area of the amplification region 26.To that end, certain sections may be thinner or thicker than othersections and/or be made from materials with different porosities,capillary forces and retention volumes. In another embodiment, the wasteregion 28 may also include a migration modifier to facilitate slower orfaster flow to control timing of valve opening.

The terminus 76 is fluidically coupled to the valve 20 such that flow tothe terminus 76 triggers opening of the valve 20. For example, the valve20 may be a chemical valve that is configured dissolve upon contact witha particular compound. In one embodiment, the valve 20 may be a hydrogelvalve configured to open at a particular pH. Flow through the terminus76 to the valve 20 at the particular pH triggers the opening. As shownin the detail views, the valve 20 separates an end 92 of the firstregion 14 from an end 94 of the second region 16. When the valve isremoved (e.g., by micromechanical actuation or chemical dissolving), thetwo ends (e., end 92 and 94) come into direct contact to fluidicallycouple and permit flow from the first region 14 to the second region 16in the direction of arrow 98. The flow may then progress through thedetection region 30 and, in certain embodiments, to a terminal pad 100.

In the depicted embodiment, the entrance 48 and the terminus 76 arepositioned relatively close together. That is, a distance between theentrance 48 and the terminus 76 may be less than a total length of awaste path through the waste region 28, e.g., the distance between theentrance 48 and the terminus 76 may be less than 50%, 25%, or 10% of thea total length of the waste path through the waste region 28. In thismanner, the entrance 48 may capture waste that is generated up to (orrelatively close to) the valve 20 to facilitate greater waste removalfrom the flow path while still providing a sufficiently long waste pathto allow the amplification reaction to progress before triggering thevalve 20.

FIG. 5 is an embodiment of the sample assessment device 10 in which thevalve 20 is fluidically separated from the terminus 76 of the wasteregion 28 (in contrast to the embodiment depicted in FIG. 4). In theembodiment of FIG. 5, the waste region 28 flows into a waste pad 110that is separated from the valve 20. The waste pad 110, when disposedproximate to the substrate 12, may also include one or more barrierlayers to prevent waste from re-entering the substrate 12. The valve 20,rather than being triggered via waste flow, is separately triggered. Forexample, in one embodiment, the valve 20 may be triggered to change froma closed to an open configuration by exposure to an environmentalstimulus (e.g., temperature, magnetic field, electric field). Theexposure may be timed to a desired amplification reaction time to permita desired amplification product generation for detection.

As shown in the exploded cross-sectional detail of the valve 20, thevalve 20 includes a hydrophilic layer and one or more hydrophobic layers124. In the valve closed configuration 130, the hydrophilic layer 120fluidically couples the end 92 of the first region 14 to the wasteregion 28, permitting lateral flow into the waste region. Thehydrophobic layer/s 124 separate both the waste region 28 and the firstregion 14 from the second region 16. In the valve open configuration132, the hydrophilic layer 120 of the valve 20 is removed to uncouplethe first region 14 from the waste region 28 (which, in turn, mayprevent backflow), and removal of one component or a portion of thehydrophobic layer 124 couples the end 92 of the first region 14 to theend 94 of the second region 16, which in turn couples the amplificationregion 26 to the detection region 30. A component or portion of thehydrophobic layer 124 remains in place after the valve 20 is open toisolate the waste region 28 from the end 94 of the second region 16 and,in turn, the detection region 30 While the depicted embodiments areillustrated with a single waste region 28, it should be understood thatthe substrate 12 may include two or more waste regions 28.

The sample assessment device 10 as provided herein may be used inconjunction with various sample application techniques. As shown in FIG.6, a capillary tube 140 may be used to apply a sample one or more sampleassessment devices 10. The capillary tube 140 includes a sample inputtube 142 forming an input passage 144. The sample input tube 142 maycouple to one or more output tubes 146 that terminate in openings 148that are designed to be positioned in contact with the sampleapplication region of the substrate 12. The substrate 12 may alsoinclude fluid application regions, e.g., amplification fluid applicationregion 150 and wash fluid application region 152. In this manner, thesubstrate 12 may have additional sample preparation, amplification, andwash functionality, depending on the type of sample. The capillary tube140 allows the same sample to be placed on multiple substrates 12substantially simultaneously for parallel assessment. In one example,the parallel assessment may include determining if different sequencesof interest are present in the sample. In another embodiment, theassessment may be redundant for quality purposes.

FIG. 7 and FIG. 8 illustrate examples of different types of biologicalsamples that may be used in conjunction with capillary tubes 140. Inparticular, depending in the type of biological sample (e.g., wholeblood vs. tissue swab), different types of capillary tubes 140 may beused to receive the sample and apply the sample to the sample assessmentdevice 10. In this manner, the sample assessment device 10 is capable ofbeing used for different types of samples by selecting the appropriatecapillary tube 140 for the desired sample. In certain embodiments, thesample assessment device 10 may be provided as a kit with one or morecapillary tubes 140. In one embodiment, the kit may include packaging ofa plurality of capillary tubes 140 specific for respective sample typestogether with one or more sample assessment devices 10. In addition, thecapillary tubes 140 may be configured to receive point-of-care samplesfor rapid analysis.

FIG. 7 is a cross-sectional view of the sample assessment device 10 andcapillary tube 140 specific for use with a sample swab 154. Thecapillary tube 140 includes a reservoir 156 proximate to the opening 144to receive the sample swab 154. The capillary tube 140 also includes aseal 158 to separate the swab and any sample preparationbuffers/reagents from the flow channel 160. When appropriate incubationtime has elapsed to allow the sample to be eluted into the reservoir,the seal 158 may be broken to allow the sample to enter the capillaryflow channel 160 and enter the sample application region 24 via theopening 148. In the depicted embodiment, the substrate includes a lyticsubstrate pad 162 for additional sample preparation. FIG. 8, incontrast, is illustrated with a capillary tube 140 specific for bloodsamples 170. In one embodiment, the blood sample 170 may be receiveddirectly from a patient (i.e., without intervening storage). Thecapillary tube 140 for the blood sample 170 does not include a reservoiror seal, and flow progresses directly to the lytic pad 162 of the sampleapplication region 24 of the substrate 12.

FIG. 9 is a flow diagram of a sample assessment method 200. Inoperation, a sample assessment device is provided (block 210) and asample is applied to the sample assessment device (block 212). Theapplication of the sample may be manual or automatic. Further, asprovided herein, the sample application may employ one or more capillarytubes (e.g., capillary tube 140, see FIG. 5). After the sample has beenapplied to the sample assessment device, the method 200 allows anamplification reaction to occur to generate amplification products(block 214). In addition, the method 200 allows waste from samplepreparation and/or amplification to flow into a waste region of thesample assessment device (block 216). After the amplification reactionhas occurred and after waste has flowed into the waste region, the valveis allowed to passively open (e.g., via waste flow that triggers tovalve) or the valve is actively triggered (e.g., activation of anenvironmental condition or application of a chemical to the valve)(block 218). When the valve is opened, the method 200 allows theamplification products to flow into the detection region (block 220).

FIG. 10 is a system 250 for detection of the amplified products in thedetection region 30. The detection region 30 refers to an area on thesample assessment device 10 configured to facilitate detection ofamplified products of the target nucleic acid sequence. In oneembodiment, the amplified products are detected using nucleic acidcapture probes immobilized on the substrate 12. The target nucleic acidin the amplified products of a sample is captured at the detectionregion 30 of the substrate 12 and thereby separated from the othernon-target materials. In some embodiments, after capturing the targetnucleic acids, detection probes are added to the substrate 12 and thecaptured nucleic acids are detected by binding with the detection probe.In some embodiments, the detection region 30 includes impregnateddetection probes. While certain embodiments of the disclosed techniquespermit one-step amplification and detection of amplified products in thedetection region 30, it should also be understood that the migratedamplified product in the detection region 30 may be stored for lateranalysis.

The capture probes may include a nucleic acid that comprises at leastone sequence that is complementary to at least one sequence of thetarget nucleic acid. In some embodiments, the nucleic acid capture probeis deoxyribonucleic acid (DNA). The nucleic acid capture probe maycomprise multiple sequences that are complimentary to the DNA sequenceof the target nucleic acid, such as a target DNA. The nucleic acidcapture probe is applied to capture zone of a substrate, wherein thecapture probe captures the target nucleic acid by hybridization.

The amplified products captured by the capture probes may be detectedusing signal molecules present in the amplified products, the captureprobes, or primary and/or secondary detection probes. The signalgenerators may include a chromophore moiety, a fluorescent moiety, aphosphorescence moiety, an affinity probe, a magnetic probe, aparamagnetic probe, a plasmonic nanoparticle probe or combinationsthereof. The primary detection probe may further comprise a bindingmoiety such as a biotin or an antibody, a streptavidin, a gold particleor combinations thereof.

The detection system 250 includes one or more sensors or detectors 252for detecting the signal generated by the detection zone and coupled toan assessment device 254. The detector 252 may be configured to detectone or more signal molecules. For example, in certain embodiments, thedetector 252 may be configured to detect optical or electrochemicalsignals. The sample assessment device 10 may couple to the detector 252to facilitate detection at one or more locations along the substrate 12.The system 250 may also include heating elements 256 for heating thesample assessment device 10 to activate amplification and/or to open thevalve. For example, the amplification reagents may be configured toreact at particular temperatures or using particular temperature cycles.Accordingly, the system 250 may be configured to provide cyclical orisothermal heating to the substrate 12. In addition, the valve may beconfigured to open at particular temperatures. The heating elements 256may be configured to open the valve after an a predetermined amount oftime for amplification passes or after a predetermined amount of timeafter sample application, e.g., based on calibration or empiricalresults to achieve sufficient amplification.

The system 250 may also include computer-readable memory circuitry 258,such as magnetic, electronic, or optical storage media, for storingprograms and routines executed by the device 254. The stored programs orroutines may include programs or instructions for performing all or partof the present techniques. The system 250 may also include processingcircuitry 260 for executing the programs or instructions, e.g.,instructions to open the valve. The device 254 may also include variousinput/output (I/O) interfaces 262, as well as various network orcommunication interfaces to allow communication with user interfacedevices, such as a display 264, that may be used for viewing andinputting quantification information. The various network andcommunication interfaces may allow connection to both local and widearea intranets and storage networks as well as the Internet. The variousI/O and communication interfaces may utilize wires, lines, or suitablewireless interfaces, as appropriate or desired.

Technical effects of the disclosed embodiments include rapid nucleicacid amplification and assessment. That is, the disclosed techniquesfacilitate amplification and detection in a single step.

This written description uses examples, including the best mode, andalso to enable any person skilled in the art to practice the disclosedembodiments, including making and using any devices or systems andperforming any incorporated methods. The patentable scope is defined bythe claims, and may include other examples that occur to those skilledin the art. Such other examples are intended to be within the scope ofthe claims if they have structural elements that do not differ from theliteral language of the claims, or if they include equivalent structuralelements with insubstantial differences from the literal languages ofthe claims.

1. A method of assessing a sample comprising: providing a sampleassessment device comprising: a sample application region comprising aplurality of lysis reagents; an amplification region capable of beingheated to a temperature range sufficient for nucleic acid amplification;a waste region comprising an entrance coupled to the amplificationregion and extending away from the amplification region; a detectionregion spaced apart from the amplification region; and a valveseparating the amplification region from the detection region; applyinga sample comprising nucleic acids to the sample application region;allowing the sample to react with the lysis reagents in a lysisreaction; allowing the sample to flow into the amplification region andreact with amplification reagents for a duration of time in anamplification reaction to generate amplification products of a nucleicacid sequence of interest; allowing waste from the sample and lysisreaction to flow into the waste region; and allowing the amplificationproducts to flow through the valve to enter the detection region,wherein the valve is configured to be closed during the amplificationreaction and open after the duration of time.
 2. The method of claim 1,comprising applying a liquid on the sample assessment device to flowfrom the sample application region to the amplification region and thewaste region.
 3. The method of claim 1, comprising detecting the nucleicacid sequence of interest in the detection region.
 4. The method ofclaim 3, wherein the detection region comprises nucleic acid captureprobes complementary to the nucleic acid sequence of interest andwherein the detection is based on detecting amplification products thathave hybridized to the capture probes.
 5. The method of claim 4, whereinthe nucleic acid capture probe comprises deoxyribonucleic acids (DNA),ribonucleic acids (RNA), nucleotide analogues, or combinations thereof.6. The method of claim 1, wherein the sample amplification region andthe amplification region are at least partially overlapping on thesubstrate.
 7. The method of claim 1, wherein the amplification reagentsare impregnated in the substrate
 8. The method of claim 1, comprisingapplying the amplification reagents to the substrate.
 9. The method ofclaim 7, wherein the lysis reagents flow into the waste region such thatan amount of lysis reagent present during amplification is reduced. 10.The method of claim 1, wherein the sample application region and theamplification region are fluidically isolated from the detection regionwhen the valve is closed.
 11. The method of claim 1, wherein the wasteregion is fluidically isolated from the detection region when the valveis closed.
 12. The method of claim 1, wherein the valve comprises ahydrogel valve.
 13. The method of claim 1, wherein the valve isconfigured to dissolve or melt to open.
 14. The method of claim 1,comprising allowing flow in the waste region to extend to a terminus ofthe waste region, wherein the terminus of the waste region is coupled tothe valve such that the flow to the terminus triggers opening of thevalve.
 15. The method of claim 1, comprising triggering the valve toopen via application of a chemical reagent or an environmental stimulus.16. A sample assessment device comprising: a substrate, wherein thesubstrate comprises: a sample application region; an amplificationregion; a waste region comprising an entrance fluidically coupled to theamplification region and extending away from the amplification region;and a detection region spaced apart from the amplification region; and avalve coupled to the substrate and configured to separate theamplification region from the detection region in a closedconfiguration, wherein the amplification region and the valve arepositioned on the sample assessment device between the sampleapplication region and the detection region and wherein the sampleassessment device is configured to permit lateral flow from theamplification region to the detection region when the valve is in anopen configuration.
 17. The device of claim 16, wherein the waste regionextends away from an elongated strip, wherein the sample applicationregion, the amplification region, and the detection region arepositioned on the elongated strip.
 18. The device of claim 16, whereinthe waste region comprises a first portion orthogonal to a flow axis ofthe elongated strip and a second portion parallel to the flow axis ofthe elongated strip.
 19. The device of claim 16, wherein the wasteregion comprises a plurality of portions arranged orthogonally to oneanother.
 20. The device of claim 16, wherein the entrance and a terminusof the waste region are separated by a distance that is less than atotal length of a flow path of the waste region.
 21. The device of claim20, wherein the distance is less than 10% of the total length of theflow path of the waste region.
 22. The device of claim 16, wherein atotal surface area of the waste region is configured to be greater thana total surface area of the amplification region.
 23. The device ofclaim 16, wherein the amplification region comprises amplificationreagents comprise one or more primers complementary to the nucleic acidsequence of interest.
 24. The device of claim 16, comprising a sampleapplication tube coupled to the substrate and configured to receive asample and apply the sample to the sample application region.
 25. Thedevice of claim 16, wherein the valve comprises a hydrogel valveresponsive to glucose, a magnetic field, or temperature.
 26. The deviceof claim 16, wherein the sample assessment device comprises a cellulosemembrane, a nitrocellulose membrane, a modified porous nitrocellulose orcellulose based membrane, a poly(ethyleneglycol)-modifiednitrocellulose, a cellulose acetate membrane, a nitrocellulose mixedester membrane, a glass fiber, a quartz fiber, a polyethersulfonemembrane, a nylon membrane, a polyolefin membrane, a polyester membrane,a polycarbonate membrane, a polypropylene membrane, a polyvinylidenedifluoride membrane, a polyethylene membrane, a polystyrene membrane, apolyurethane membrane, a polyphenylene oxide membrane, apoly(tetrafluoroethylene-co-hexafluoropropylene) membrane, or acombination thereof.
 27. The device of claim 16, comprising lysisreagents disposed in the sample application region.
 28. The device ofclaim 16, comprising amplification reagents disposed in theamplification region.
 29. The device of claim 16, wherein theamplification region is at least partially overlapping with the sampleapplication region.
 30. A sample assessment system comprising: asubstrate comprising: a first region comprising a plurality of lysisreagents; a second region comprising an entrance fluidically coupled tothe first region and extending away from the first region; and a thirdregion spaced apart from the first region; a valve separating the firstregion from the third region in a closed configuration to preventlateral flow from the first region to the third region and configured topermit the lateral flow in an open configuration; a detector configuredto detect one or more signal molecules in the substrate and provide anoutput; and a processor configured to receive the output and determine apresence or concentration of a nucleic acid sequence of interest in thebiological sample based on the output.
 31. The system of claim 30,comprising a heater positioned to heat the first region during anamplification reaction; and a controller coupled to the heater andconfigured to control the activation and temperature of the heater. 32.The system of claim 30, wherein the first region comprises anamplification region and lysis region spaced apart from one another. 33.The system of claim 30, wherein the first region comprises anamplification region and lysis region that are at least partiallyoverlapping
 34. The system of claim 31, wherein the heater comprises aportion positioned to heat the valve to trigger the open configurationof the valve.